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Keywords:

  • IGFBP-2 suppression;
  • growth inhibition;
  • prostate cancer.

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. References

Background:  Insulin-like growth factor binding protein-2 (IGFBP-2) is expressed by all human prostate cancer cell lines and dramatically increases in the serum of prostate cancer patients. However, the role of IGFBP-2 in prostatic tumorigenesis is not known. The aim of the present study was to investigate the effects of IGFBP-2 on the proliferation of DU145 human prostate cancer cells in culture.

Methods:  Using cell proliferation assays, we examined the effects of exogenously administered and endogenously modulated levels of IGFBP-2 on the proliferation of DU145 cells.

Result:  Cell growth was stimulated by exogenously administered IGFBP-2, but significantly retarded (P < 0.05) by its neutralizing antibody. Overexpression of IGFBP-2 by transfection also stimulated cell growth, which was significantly (P < 0.05) inhibited in transfectants expressing antisense mRNA to IGFBP-2. Furthermore, the proliferation of IGFBP-2 overexpressing cells was significantly dampened by exogenously administered IGFBP-2 antibody.

Conclusions:  IGFBP-2 is an autocrine growth factor for DU145 human prostate cancer cells and cell proliferation can be significantly retarded by neutralizing or inhibiting its synthesis. These findings provide a strong rationale for targeting IGFBP-2 in the testing of novel strategies to treat prostate cancer.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. References

Prostate cancer is the second leading cause of cancer-related deaths in men in the USA.1 Despite a variety of treatment options ranging from surgery to systemic cytotoxic therapy, there is no curative therapy for metastatic prostate cancer to date. Therefore, the identification of unique, or preferentially expressed, gene products in prostate cancer cells and tissues is of utmost importance for the development and evaluation of novel treatment strategies.

A variety of growth factors, including the insulin-like growth factor (IGF) system have been implicated in the regulation of prostate tumor cell growth. The IGF system is composed of two peptide ligands (IGF-I and IGF-II), two receptors (IGF-IR and IGF-IIR), and at least six high-affinity IGF-binding proteins (IGFBP-1 to IGFBP-6). Protein and mRNA for the ligands, receptors and binding proteins 2 through 6 are present in normal and malignant prostate epithelial and stromal cells.2,3

The IGFBP modulate the bioavailability of IGF to their receptors.4 More recently, IGF-independent actions of IGFBP have been recognized,5,6 suggesting that IGFBP are a separate class of growth modulators. In the prostate, the major binding protein is IGFBP-2. Prostate cancer patients have two to three times higher than normal levels of serum IGFBP-27–9 and the elevation is directly correlated with the tumor Gleason score and serum prostate-specific antigen (PSA) levels.8,9 However, the role of IGFBP-2 in the development and/or progression of prostate cancer is little known.

In the present study, we evaluated the effects of IGFBP-2 on the proliferation of DU145 human prostate cancer cells. We first examined the effects of exogenous IGFBP-2 or its neutralizing antibody on cellular proliferation. We then stably transfected the cells with IGFBP-2 or anti-IGFBP-2 expression vectors to modulate the endogenous levels of IGFBP-2 and analyzed the effects on cell proliferation. We show evidence that proliferation of DU145 cells is modulated by both exogenous and endogenous IGFBP-2 and that cell growth is significantly retarded by suppression of this protein.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. References

Cells and cell culture

Human prostate tumor cell line DU145, was purchased from American Type Culture Collection (Rockville, MD). Cells were cultured in Dulbeco's Modified Eagle Medium (DMEM; Life Technologies, Grand Island, NY) supplemented with 10% fetal calf serum (FCS; Hyclone, Logan, UT), 1% penicillin and streptomycin and 0.05% amphotericin B at 37°C in a humidified atmosphere containing 95% air and 5% CO2. Cells were maintained at subconfluent densities by subculturing stock cultures at appropriate intervals.

Proteins and other reagents

Recombinant human IGFBP-2, polyclonal antibody (IGFBP-2 Ab) and an antibody to human IGF-IR α subunit were purchased from Research Diagnostics, Inc (Flanders, NJ). All other reagents were from Sigma (St. Louis, MO), unless otherwise stated.

Cell proliferation bioassays

Growth of prostate cancer cells in response to IGFBP-2, IGFBP-2 Ab and other IGF system components was measured by counting the total number of cells at the end of the experimental period and/or by the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI).

Cell counting and cell treatment

Cells were seeded in 60-mm dishes (4 × 104 cells/dish) in 10% FCS supplemented DMEM. They were allowed to attach overnight, serum starved for 24 h and then treated with 125, 250 or 500 ng of IGFBP-2 or anti-IGFBP-2 immunoglobulin G (IgG) per mL for 72 h. The doses of IGFBP-2 administered were based on the serum levels of the protein in normal (367 ± 44 µg/L) and prostate cancer patients (560 ± 66 µg/L) and were within the normal physiological range. After treatment, the cells were dispersed using trypsin-EDTA (1X) solution and counted using a hemocytometer. More than 98% of the cells were viable, as determined by trypan blue exclusion. The effect of non-immune serum IgG on cell growth was also determined.

MTS cell proliferation assay

Cells were seeded in 96-well plates (6 × 102 cells/well) in 10% FCS supplemented DMEM and allowed to attach overnight. The cell monolayers were incubated in serum-free media for 24 h, and then treated as described in the preceding paragraph. Cell proliferation was measured by the Celltiter 96 Aqueous One Solution Cell Proliferation Assay (Promega) according to the manufacturer's protocol. Upon color conversion, samples were read at optical density 490 nm on a scanning multiwell spectrophotometer (Vmax and SOFTmax software, Molecular Devices, Menlo Park, CA). The optical absorbance values for test wells were obtained by subtracting the blank (cell-free wells containing culture media only).

Clonogenic assay

Cells (2 × 104 cells/mL) were suspended in 0.3% Sea Plaque agarose containing DMEM (supplemented with 10% FCS, insulin, transferrin and selinium) and overlaid on to a basal layer of 0.5% agarose in 6 well plates. The cultures were administered increasing concentrations of IGFBP-2 and/or anti-IGFBP-2 IgG and maintained in a humidified atmosphere at 37°C for 5 weeks to facilitate colony formation. Live colonies were stained with 0.3% methylene blue, counted (>1 mm in size) and photographed using an inverted light microscope (Olympus, Houston, TX).

Generation of stable IGFBP-2 sense and antisense constructs

Sense (S) and Antisense (AS) constructs were generated by cloning a 1.1 kilobase full-length IGFBP-2 cDNA (a gift from Dr David Powell, Baylor College of Medicine, Houston, TX) into pcDNA 3.1 (+/–), containing a neomycin (G418) resistance gene (Invitrogen, Carlsbad, CA). Directional cloning of the cDNA inserts was confirmed by restriction mapping. DU145 cells were transfected with the constructs using Lipofectamine Plus Reagent (Promega). Control (C) clones were generated by transfecting cells with empty vectors. The transfected cells were maintained in media containing G418 (1 mg/mL). Antibiotic resistant S, AS and their respective C clones were selected by limiting dilution (16–18 clones per group) and each was expanded further under continued drug selection.

Western blot analysis of IGFBP-2

As IGFBP-2 is a secreted protein, expression was measured in both cell lysates and conditioned media (CM). Samples of CM were collected from serum-starved cultures in the presence of a cocktail of protease inhibitors containing 4-(2-aminoethyl)benzenesulfonyl fluoride, pepstatin A, trans-epoxysuccinyl-L-leucyl-amido(4-guanidino)butane (E-64), bestatin, leupeptin and aprotinin (Catalog ♯P8340, Sigma). The samples were concentrated using Centriplus YM (10 000 molecular weight cutoff ) centrifugal filters (Millipore Inc., Bedford, MA), according to the manufacturer's instructions. Lysates were prepared from cells expressing high or low levels of IGFBP-2 using Laemmli sample preparation buffer.

Conditioning media samples and cell lysates from an equivalent number of cells were separated by 12% non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), following standard procedures.10 Proteins were electrotransferred onto polyvinnylidene difluoride membranes, which were blocked with 5% non-fat dry milk/Tris-buffered saline with 0.1% (v/v) Tween 20 for 1 h at 22°C. Membranes were incubated overnight in goat antihuman IGFBP-2 antibody (1 : 250 dilution; Research Diagnostics Inc, Flanders, NJ) at 4°C and then with a horseradish peroxidase-labeled secondary antibody for 1 h at 22°C. Immunoreactive proteins were detected by enhanced chemiluminescence Western blotting detection system (Amersham Pharmacia, Arlington Heights, IL) as described in the product manual.

Densitometric analysis

The relative expression of IGFBP-2 following SDS-PAGE and Western blotting was analyzed by densitometric measurements, using the Chemi Imager 4400 Low Light Imaging System (Alpha Innotech Corp., San Leandro, CA).

Statistical analysis

Data for growth assays were expressed as mean ± standard error. Statistical significance was analyzed by using Student's t-test and analysis of variance (anova). Differences were regarded as significant when P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. References

Effect of IGFBP-2 on DU145 cell proliferation

We tested the influence of IGFBP-2 and its Ab (125, 250 and 500 ng/mL) on the proliferation of DU145 cells following 72 h of treatment. Proliferation was examined by counting cell numbers and by the Cell proliferation assay (Promega). Treatment resulted in a significant increase in cell proliferation with IGFBP-2 and decrease with the Ab in a dose-dependent manner when compared to the growth of untreated cells (Fig. 1a, b). Equivalent concentrations of non-immune IgG, however, had no effect on cell proliferation (Fig. 1c).

image

Figure 1. Effect of insulin-like growth factor binding protein (IGFBP)-2 and its antibody (Ab) on the proliferation of DU145 cells. (a) Changes in cell numbers following treatment with IGFBP-2 and IGFBP-2 Ab. Cells were treated with IGFBP-2 or its Ab for 72 h in serum-free media. Viable cells identified by the trypan blue exclusion method were counted under the microscope. Values shown are mean ± standard error of triplicate samples from three separate experiments. (b) Cell proliferation assay to examine the effect of IGFBP-2 and Ab on DU145 cell growth. Cells seeded in 96-well plates were treated with IGFBP-2 or its antibody for 72 h in serum-free media and growth was measured by the Celltiter 96 Aqueous One Solution Cell Proliferation Assay, as per kit instructions. Results presented are mean ± standard error of triplicate samples from three separate experiments. (c) Effect of non-immune immunoglobulin G (IgG) on the proliferation of DU145 cells. Cells were seeded in 96-well plates as described above and proliferation was measured following treatment with non-immune IgG for 72 h. Results presented are mean ± standard error of quadruplicate samples from three separate experiments. ▪, serum-free; bsl00039, 125 ng/mL; bsl00052, 250 ng/mL; bsl00068, 500 ng/mL. *, P < 0.05 by Student's t-test.

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Since synergistic mechanisms have been postulated between IGFBP-2 and IGF,11 we evaluated the dependence of IGFBP-2 on IGF-I receptor (R) for modulating cell growth by treating cultures of DU145 cells with IGF-IR Ab or IGF-IR Ab + IGFBP-2 for 72 h. Growth was significantly retarded by IGF-IR Ab at all three doses tested, but cell proliferation was significantly improved by coadministration of 125 or 250 ng/mL of IGFBP-2 (Fig. 2).

image

Figure 2. Growth-modulating effects of insulin-like growth factor (IGF)-IR antibody (Ab) and insulin-like growth factor binding protein (IGFBP)-2 on DU145 cells. Cells were seeded at 6 × 102/well in 96-well plates and proliferation was measured following treatment of the cells with IGF-IR Ab singly or in combination with increasing concentrations of IGFBP-2 for 72 h. Results presented are mean ± standard error of triplicate samples from three separate experiments. ▪, serum-free; bsl00039, 125 ng/mL; bsl00052, 250 ng/mL; bsl00068, 500 ng/mL. *, P < 0.05 by Student's t-test.

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Influence of IGFBP-2 and its Ab on DU145 colony formation

We tested the influence of IGFBP-2 and its Ab on the clonogenic potential of DU145 cells using a soft-agar assay. Microphotographs of colonies formed in the culture plates after 5 weeks of culture are shown in Fig. 3a. The total numbers of colonies (>1 mm) formed in the culture plates were counted. The number of colonies formed was consistently higher than the control with increasing doses of IGFBP-2, but decreased in a dose–dependent fashion in cultures treated with IGFBP-2 Ab (Fig. 3b).

image

Figure 3. (a) Photomicrographs of soft-agar colonies of DU145 cells treated with insulin-like growth factor binding protein (IGFBP)-2 or its antibody (Ab). Cells were administered IGFBP-2 or IGFBP-2 Ab and seeded at 2 × 104/well in 6-well plates in 0.3% agarose-containing Dulbeco's Modified Eagle Medium supplemented with 10% Fetal Calf Serum insulin-transferrin-selenium. Colonies were allowed to form for 5 weeks. Magnification ×100; Scale bar, 200 µm for all photomicrographs. (b) Number of colonies following treatment of cultures with IGFBP-2 or its Ab. Colonies (>1 mm) formed at the end of the 5-week period were counted under a phase-contrast microscope. ▪, 10% Fetal Calf Serum; bsl00039, 125 ng/mL; bsl00052, 250 ng/mL; bsl00068, 500 ng/mL. *, P < 0.05 by Student's t-test.

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IGFBP-2 expression by transfectants

The functionality of the S, AS and corresponding C clones generated in transfection studies was determined by measuring IGFBP-2 expression in CM samples. Densitometric analysis of the immunoblots revealed that the relative concentrations of IGFBP-2 in the CM of at least four S clones (S1, 2, 13 and 16) was two to three times higher than the IGFBP-2 concentrations of the respective C clones. On the other hand, the relative concentrations of IGFBP-2 in CM of AS clones 1, 3, 4, 6 and 15 were two to four times less than the IGFBP-2 concentrations of the respective C clones. The differences in expression levels of IGFBP-2 were further confirmed by analyzing cell lysates of clones expressing higher or lower levels of IGFBP-2 in the CM. Concentrations of IGFBP-2 in the cell lysates directly correlated with the levels of IGFBP-2 secreted into the CM (Fig. 4).

image

Figure 4. Insulin-like growth factor binding protein (IGFBP)-2 protein expression and proliferation of IGFBP-2 sense-transfected (S) or antisense-transfected (AS) DU145 cells. Conditioned media (CM) was collected from serum-starved cell cultures of transfected clones and cell lysates were prepared from those clones expressing high or low levels of IGFBP-2 in the CM. Samples corresponding to 3 × 105 cells (CM) and 18 × 105 (cell lysate) were used to measure IGFBP-2 expression by Western blotting using an anti-IGFBP-2 polyclonal antibody. A 32-kDa band corresponding to IGFBP-2 was detected in all samples analyzed. (a) Densitometric analysis of the intensity of the bands revealed that IGFBP-2 concentrations in the CM (top) and cell lysates (bottom) of S clones numbered 13, 16, 2 and 1 were significantly higher than the respective Control (C) clones. Proliferation of IGFBP-2 overexpressing S clones and their respective C (empty-vector transfected) cells was measured after maintaining the cells in 2.5% Fetal Calf Serum (FCS) supplemented media for 72 h. (b) Densitometric analysis of the intensity of the bands showed that IGFBP-2 concentration in the CM (top) and cell lysates (bottom) of AS clones numbered 1, 3, 6, 15 and 4 was significantly less than the respective C clones. Proliferation of AS clones expressing low levels of IGFBP-2 and their respective C cells were measured after maintaining the cells in 2.5% FCS supplemented media for 72 h. Results presented are mean ± standard error of triplicate samples from three separate experiments. *, P < 0.05 by Student's t-test.

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Influence of modulating endogenous IGFBP-2 on proliferation of DU145 transfectants

The proliferation of representative S, AS and respective C clones was examined by the MTS assay after growing the cells in 2.5% FCS for 72 h in 96 well plates. IGFBP-2 overexpressing S clones 1, 2, 13 and 16 had significantly higher proliferative activity than the C clones. In contrast, AS clones 1, 3, 4, 6 and 15, expressing reduced levels of IGFBP-2, had significantly lower proliferative activity than the respective C clones or non-transfected cells (Fig. 4).

To test whether the growth-potentiating effects of experimentally  up-regulated  levels  of  IGFBP-2  could  be reduced by exogenously neutralizing the protein, IGFBP-2 overexpressing S clones 3, 5 and 8 were cultured in the presence of IGFBP-2 Ab for 72 h and growth was measured by the MTS assay. The growth rates of S cells were significantly retarded following treatment with 250 and 500 ng/mL of IGFBP-2 Ab (Fig. 5).

image

Figure 5. Effect of insulin-like growth factor binding protein (IGFBP)-2 antibody (Ab) on the proliferation of IGFBP-2 transfected DU145 cells. Expression of IGFBP-2 protein in the conditioned media (top) and cell lysates (bottom) was examined in clones of IGFBP-2 overexpressing Sense (S) cells and their respective Controls (C). Proliferation of S and C cells was measured after treating the cells with IGFBP-2 Ab for 72 h. Results presented are mean ± standard error of triplicate samples from three separate experiments. ▪, untreated; bsl00039, IGFBP-2 Ab 250 ng/mL; bsl00052, IGFBP-2 Ab 500 ng/mL. *, P < 0.05 by Student's t-test.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. References

Insulin-like growth factor binding proteins are potent modulators of the mitogenic effects of IGF.5 Levels of circulating IGFBP and IGFBP mRNA in the liver are highly regulated by metabolic, hormonal and developmental factors. Serum IGFBP-2 levels are dramatically elevated in a variety of human malignancies, including neuroblastoma,12 epithelial ovarian carcinomas13 and in prostate cancer;7–9 however, the role of IGFBP-2 in tumorigenesis is not understood.

To investigate the effect of IGFBP-2 on the proliferation of prostate cancer cells, we selected the aggressive, androgen-independent DU145 human prostate cancer cells, which express IGFBP-2.2 We found that exogenously administered IGFBP-2 significantly stimulated cell proliferation (250–500 ng/mL, optimal dose), while its neutralizing antibody significantly (P < 0.05) retarded cell growth in a dose-dependent manner. Non-immune serum IgG at similar concentrations had no effect on cell growth, thereby suggesting that the growth-modulating effects are specific to IGFBP-2.

We performed a soft agar assay to test the effect of IGFBP-2 and its Ab in modulating the clonogenic potential of DU145 cells. Comparing the numbers and sizes of colonies formed in each dish after 5 weeks of culture, we found that IGFBP-2 enhanced, while the antibody retarded colony formation in a dose-dependent manner.

Since the effects of exogenously administered proteins may not always mimic in vivo actions, we examined the functional consequences of modulating the expression of endogenous IGFBP-2 in DU145 cells. The present results demonstrate that the rate of cell proliferation directly correlated with the levels of IGFBP-2 expressed by the transfected cells. These findings, together with the results on exogenously administered IGFBP-2, strongly suggest that IGFBP-2 is an autocrine growth factor for DU145 cells. While the present study was in progress, Moore et al.14 demonstrated that IGFBP-2 stimulates proliferation of prostate cancer cells.

In addition to the growth stimulatory effects of IGFBP-2, the results show, for the first time, that the growth of DU145 prostate cancer cells can be significantly retarded by neutralizing IGFBP-2 with its Ab or by suppressing its expression. The growth inhibiting potential of IGFBP-2 Ab is further strengthened by our findings that the proliferation of IGFBP-2 overexpressing S cells could also be significantly retarded by the neutralizing Ab. Suppression of IGFBP-2 has also been shown to inhibit the growth of glioma cells.15

Synergistic mechanisms have been postulated between IGFBP-2 and the IGF in studies where IGFBP-2 expression correlated with a proliferative state of cells.11 Thus, after establishing the role of IGFBP-2 in modulating the growth of DU145 cells, we queried if IGFBP-2 was dependent on IGF-IR for modulating cell proliferation. The present results show that although cell proliferation was significantly inhibited by blocking IGF-IR, the proliferative activity was significantly improved following the administration of IGFBP-2 to IGF-IR Ab treated cultures. These results suggest that IGFBP-2 did not act through the IGF-IR, but rather through other pathways that can overcome inhibition of IGF signaling. This mechanism of action is not unexpected, considering the fact that IGF-IR is actually down-regulated in prostate cancer.16

To date, the precise mechanism by which IGFBP-2 manifests its effects on DU145 cells remains largely unknown. As in IGFBP-1, the Arginine-Glycine-Asparagine (RGD) integrin recognition sequence is also present in IGFBP-2.17 Non-IGF-mediated effects resulting from the interaction of IGFBP-2 with the α5β1 integrin receptor have been demonstrated for Ewing's sarcoma A673 and breast cancer cell line Hs578T.17 Whether similar interactions between IGFBP-2 with the α5β1 integrin receptors are responsible for modulating growth of DU145 cells is not known at this time. It is also not known if IGFBP-2 exerts its effects on DU145 cells through specific membrane-associated proteins or receptors, as has been demonstrated for IGFBP-3.18 However, exogenously administered IGFBP-2 translocates to the nucleus within 2 h in prostate cancer cells, suggesting that the proliferative effects of IGFBP-2 may be manifested by interactions between IGFBP-2 and non-IGF-related proteins in the nucleus14 as has been demonstrated for IGFBP-5.19 Another theoretically possible mechanism by which IGFBP-2 may promote growth is by proteolytic degradation of antitumorigenic IGFBP-3,20 because circulatory levels of IGFBP-3 are found to decrease with rising levels of IGFBP-2 in prostate cancer patients.9 Thus, additional studies are necessary to determine the specific mechanism by which IGFBP-2 modulates growth of prostate cancer cells.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. References

The results of the current investigation demonstrate that IGFBP-2 is a potent autocrine growth factor for DU145 prostate cancer cells. More importantly, the proliferation and clonogenic potential of these cells can be significantly retarded by neutralizing or suppressing the expression of IGFBP-2. These findings provide a strong basis for targeting IGFBP-2 expression at the cellular level for developing novel strategies for prostate cancer treatment.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. References

We thank Dr David Powell for providing the PKG plasmids containing the full-length human IGFBP-2 cDNA. This project was supported by American Cancer Society Institutional Research Grant 2008–99C and National Institutes of Health Grant 1 K01 AG00963–01A1 to Dr Shilla Chakrabarty (Chatterjee). A portion of this study, entitled ‘Proliferation of DU145 human prostate cancer cells is differentially modulated by IGF system components’, was presented at the Program of the 82nd Annual meeting of the Endocrine Society, Denver, CO, 2001.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Acknowledgments
  9. References
  • 1
    Landis SH, Murray T, Bolden S, Wingo PA. Cancer Statistics. Cancer J. Clinicians 1999; 49: 813.
  • 2
    Kimura G, Kasuya J, Giannini S et al. Insulin-like growth factor (IGF) system components in human prostate cancer cell lines: LNCap DU145, and PC-3 cells. Int. J. Urol. 1996; 3: 3946.
  • 3
    Thrasher JB, Tennant MK, Twomey PA, Hansberry KL, Wettalaufer JN, Plymate SR. Immunohistochemical localization of insulin-like growth factor binding proteins 2 and 3 in prostate tissue: clinical correlations. J. Urol. 1996; 155: 9991003.
  • 4
    Jones JI, Clemmons DR. Insulin-like growth factors and their binding proteins: biological actions. Endocr. Rev. 1995; 16: 334.
  • 5
    Oh Y, Yamanka Y, Wilson E, Kim HS, Vorwerck P, Hwa V. IGF-independent actions of IGFBPs. In: Takano K, Hizuka N, Takahashi SI (eds). Molecular Mechanisms to Regulate the Activities of Insulin-Like Growth Factors. Elsevier, Amsterdam 1998, 12333.
  • 6
    Perks CM, Bowen S, Gill ZP, Newcomb PV, Holly JM. Differential IGF-independent effects of insulin-like growth factor binding proteins (1–6) on apoptosis of breast epithelial cells. J. Cell. Biochem. 1999; 75: 65264.
  • 7
    Cohen P, Peehl DM, Stamey TA, Wilson KF, Clemmons DR, Rosenfeld RG. Elevated levels of insulin-like growth factor binding protein-2 in the serum of prostate cancer patients. J. Clin. Endocrinol. Metab. 1993; 76: 10315.
  • 8
    Figueroa JA, Deraad S, Tadlock L, Speights VO, Rinchart JJ. Differential expression of insulin-like growth factor binding proteins in high versus low Gleason score prostate cancer. J. Urol. 1998; 159: 137983.
  • 9
    Kanety H, Madjar Y, Dagan Y et al. Serum insulin-like growth factor binding protein-2 (IGFBP-2) is increased and IGFBP-3 is decreased in patients with prostate cancer: correlation with serum prostate-specific antigen. J. Clin. Endocrinol. Metab. 1993; 77: 22933.
  • 10
    Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 6805.
  • 11
    Hoeflich A, Lahm H, Blum W, Kolb H, Wolf E. Insulin-like growth factor-binding protein-2 inhibits proliferation of human embryonic kidney fibroblasts and of IGF-responsive colon carcinoma cell lines. FEBS Letters 1998: 32934.
  • 12
    Menouny M, Binoux M, Babajko S. Role of insulin-like growth factor binding protein-2 and its limited proteolysis in neuroblastoma cell proliferation: modulation by transforming growth factor-beta and retinoic acid. Endocrinology 1997; 138: 68390.
  • 13
    Kanety H, Kattan M, Goldberg I et al. Increased insulin-like growth factor binding protein-2 (IGFBP-2) gene expression and protein production lead to high IGFBP-2 content in malignant ovarian cyst fluid. Br. J. Cancer 1996; 73: 106973.
  • 14
    Moore MG, Wetterau LA, Francis MJ, Peehl DM, Cohen P. Novel stimulatory role for insulin-like growth factor binding protein-2 in prostate cancer cells. Int. J. Cancer 2003; 105: 14–19.
  • 15
    Wang ZH, Ma J, Zeng BJ et al. Correlation of glioma cell regression with inhibition of insulin-like growth factor 1 and insulin-like growth factor binding protein-2 expression. Neuroendocrinology 1997; 66: 20311.
  • 16
    Tennant MK, Thrasher JB, Twomey PA, Drivdahl RH, Plymate SR. Protein and messenger ribonucleic acid (mRNA) for the type 1 insulin-like growth factor (IGF) receptor is decreased and IGF-II mRNA is increased in human prostate carcinoma compared to benign prostate epithelium. J. Clin. Endocrinol. Metab. 1996; 81: 377482.
  • 17
    Hoeflich A, Reisinger R , Lahm H et al. Insulin-like growth factor-binding protein 2 in tumorigenesis: Protector or promoter? Cancer Res. 2001; 61: 860110.
  • 18
    Oh Y, Muller HL, Pham H, Rosenfeld RG. Demonstration of receptors for insulin-like growth factor binding protein-3 on Hs578T human breast cancer cells. J. Biol. Chem. 1993; 268: 260458.
  • 19
    Schedlich LJ, Young TF, Firth SM, Baxter RC. Insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5 share a common nuclear transport pathway in T47D human breast carcinoma cells. J. Biol. Chem. 1998; 273: 1834752.
  • 20
    Menouy M, Binoux M, Babajko S. IGFBP-2 expression in a human cell line is associated with increased IGFBP-3 proteolysis, decreased IGFBP-1 expression and increased tumorigenecity. Int. J. Cancer 1998; 77: 8749.